The present invention relates to a process for producing a dipeptide which comprises culturing in a medium a microorganism which has the ability to produce a protein having the activity to form the dipeptide from one or more kinds of amino acids and which has the ability to produce at least one of said one or more kinds of amino acids, allowing the dipeptide to form and accumulate in the medium, and recovering the dipeptide from the medium.
At present, many of the amino acids are produced by the so-called fermentation method (Hiroshi Soda, et al., Amino Acid Fermentation, Gakkai Shuppan Center (1986) and Biotechnology 2nd ed., Vol. 6, Products of Primary Metabolism, VCH Verlagsgesellschaft mbH, Weinheim (1996)). The fermentation method as used herein refers to a method in which a microorganism is cultured in a medium comprising inexpensive substances such as glucose, acetic acid, methanol, ammonia, ammonium sulfate and corn steep liquor to obtain a desired amino acid by utilizing the metabolic activity of the microorganism. The fermentation method is excellent as a method for producing amino acids from inexpensive materials with light burdens on the environment.
As for the method for large-scale peptide synthesis, chemical synthesis methods (liquid phase method and solid phase method), enzymatic synthesis methods and biological synthesis methods utilizing recombinant DNA techniques are known. Currently, the enzymatic synthesis methods and biological synthesis methods are employed for the synthesis of long-chain peptides longer than 50 residues, and the chemical synthesis methods and enzymatic synthesis methods are mainly employed for the synthesis of dipeptides.
In the synthesis of dipeptides by the chemical synthesis methods, operations such as introduction and removal of protective groups for functional groups are necessary, and racemates are also formed. The chemical synthesis methods are thus considered to be disadvantageous in respect of cost and efficiency. They are unfavorable also from the viewpoint of environmental hygiene because of the use of large amounts of organic solvents and the like.
As to the synthesis of dipeptides by the enzymatic methods, the following methods are known: a method utilizing reverse reaction of protease (J. Biol. Chem., 119, 707-720 (1937)); methods utilizing thermostable aminoacyl t-RNA synthetase (Japanese Published Unexamined Patent Application No. 146539/83, Japanese Published Unexamined Patent Application No. 209991/83, Japanese Published Unexamined Patent Application No. 209992/83 and Japanese Published Unexamined Patent Application No. 106298/84); a method utilizing reverse reaction of proline iminopeptidase (WO03/010307 pamphlet); and methods utilizing non-ribosomal peptide synthetase (hereinafter referred to as NRPS) (Chem. Biol., 7, 373-384 (2000), FEBS Lett., 498, 42-45 (2001), U.S. Pat. No. 5,795,738 and U.S. Pat. No. 5,652,116).
However, the method utilizing reverse reaction of protease requires introduction and removal of protective groups for functional groups of amino acids used as substrates, which causes difficulties in raising the efficiency of peptide-forming reaction and in preventing peptidolytic reaction. The methods utilizing thermostable aminoacyl t-RNA synthetase have the defects that the expression of the enzyme and the prevention of side reactions forming by-products other than the desired products are difficult. The method utilizing proline iminopeptidase requires amidation of one of the amino acids used as substrates. The methods utilizing NRPS are inefficient in that the supply of coenzyme 4′-phosphopantetheine is necessary.
In addition to the above defects, these methods are disadvantageous in respect of production cost because all of them use amino acids or derivatives thereof as substrates.
On the other hand, there exist a group of peptide synthetases that have enzyme molecular weight lower than that of NRPS and do not require coenzyme 4′-phosphopantetheine; for example, γ-glutamylcysteine synthetase, glutathione synthetase, D-alanyl-D-alanine (D-Ala-D-Ala) ligase, and poly-γ-glutamate synthetase. Most of these enzymes utilize D-amino acids as substrates or catalyze peptide bond formation at the γ-carboxyl group. Because of such properties, they can not be used for the synthesis of dipeptides by peptide bond formation at the α-carboxyl group of L-amino acid.
It is reported that a protein bearing no similarity to NRPS (albC gene product) is responsible for the synthesis of the cyclo(L-phenylalanyl-L-leucine) structure in Streptomyces noursei ATCC 11455 known as a strain producing the antibiotic albonoursin and that albonoursin was detected when cyclo dipeptide oxidase was made to act on the culture broth of Escherichia coli and Streptomyces lividans into which the albC gene was introduced (Chemistry & Biol., 9, 1355-1364 (2002)). However, there is no report that the albC gene product forms a straight-chain dipeptide.
The only known example of an enzyme capable of dipeptide synthesis by the activity to form a peptide bond at the α-carboxyl group of L-amino acid is bacilysin (dipeptide antibiotic derived from a microorganism belonging to the genus Bacillus) synthetase. Bacilysin synthetase is known to have the activity to synthesize bacilysin [L-alanyl-L-anticapsin (L-Ala-L-anticapsin)] and L-alanyl-L-alanine (L-Ala-L-Ala), but there is no information about its activity to synthesize other dipeptides (J. Ind. Microbiol., 2, 201-208 (1987) and Enzyme. Microbial. Technol., 29, 400-406 (2001)).
As for the bacilysin biosynthetase genes in Bacillus subtilis 168 whose entire genome information has been clarified (Nature, 390, 249-256 (1997)), it is known that the productivity of bacilysin is increased by amplification of bacilysin operons containing ORFs ywfA-F (WO00/03009 pamphlet). However, it is not known whether an ORF encoding a protein having the activity to ligate two or more amino acids by peptide bond is contained in these ORFs, and if contained, which ORF encodes the protein.
That is, no method has so far been known for producing a dipeptide consisting of one or more kinds of amino acids by fermentation.
An object of the present invention is to provide a process for producing a dipeptide which comprises culturing in a medium a microorganism which has the ability to produce a protein having the activity to form the dipeptide from one or more kinds of amino acids and which has the ability to produce at least one of said one or more kinds of amino acids, allowing the dipeptide to form and accumulate in the medium, and recovering the dipeptide from the medium.